Plasmids are extrachromosomal genetic elements and are capable of autonomous replication within their hosts. Bacterial plasmids range in size from 1 Kb to 200 Kb or more and encode a variety of useful properties. Plasmid encoded traits include resistance to antibiotics, production of antibiotics, degradation of complex organic molecules, production of bacteriocins, such as colicins, production of enterotoxins, and production of DNA restriction and modification enzymes. Although plasmids have been studied for a number of years in their own right, particularly in terms of their replication, transmissibility, structure and evolution, with the advent of genetic engineering technology the focus of plasmid research has turned to the use of plasmids as vectors for the cloning and expression of foreign genetic information. In its application as a vector, the plasmid should possess one or more of the following properties. The plasmid DNA should be relatively small but capable of having relatively large amounts of foreign DNA incorporated into it. The size of the DNA insert is of concern in vectors based on bacteriophages where packing the nucleic acid into the phage particles can determine an upper limit. The plasmid should be under relaxed replication control. That is, where the replication of the plasmid molecule is not strictly coupled to the replication of the host DNA (stringent control), thereby resulting in multiple copies of plasmid DNA per host cell. The plasmid should express one or more selectable markers, such as the drug resistance markers, mentioned above, to permit the identification of host cells which contain the plasmid and also to provide a positive selection pressure for the maintenance of the plasmid in the host cell. Finally the plasmid should contain a single restriction site for one or more endonucleases in a region of plasmid which is not essential for plasmid replication. It is particularly useful if such a site is located within one of the drug resistance genes thereby permitting the monitoring of successful integration of the foreign DNA segment by insertional inactivation. For example, when a plasmid contains two drug resistance genes and one of the genes contains a single restriction endonuclease site, the foreign DNA when ligated into that site will interrupt the expression of the drug resistance gene, thus converting the phenotype of the host from double drug resistance to single drug resistance. A vector as described above is useful for cloning genetic information, by which is meant integrating a segment of foreign DNA into the vector and reproducing identical copies of that information by virtue of the replication of the plasmid DNA.
The next step in the evolution of vector technology was the construction of so-called expression vectors. These vectors are characterized by their ability not only to replicate the inserted foreign genetic information but also to promote the transcription of the genetic information into mRNA and its subsequent translation into protein. This expression requires a variety of regulatory genetic sequences including but not necessarily limited to promoters, operators, transcription terminators, ribosomal binding sites and protein synthesis initiation and termination codons. These expression elements can be provided with the foreign DNA segment as parts thereof or can be integrated within the vector in a region adjacent to a restriction site so that when a foreign DNA segment is introduced into the vector it falls under the control of those elements to which it is now chemically joined.
In a more recent development, hybrid vectors have been constructed which permit the cloning and/or expression of foreign genetic information in more than one host. These biphasic or shuttle vectors are characterized as having separate origins of replication (replicons) to permit replication of the plasmid in the desired host; further, in the case of expression vectors, it may be required to have two sets of regulatory elements, each specific for the intended host. Such duplication of regulatory elements is not always required as it may be possible for a single promoter to be able to function in both of the desired hosts. Regardless of the type of biphasic vector, be it either a cloning or expression vector, it is preferred to have at least two selectable markers, one permitting selection in each of the contemplated hosts.
Examples of biphasic vectors include: a bifunctional plasmid (pMP358) capable of cloning and expressing human dihydrofolate reductase cDNA in both Escherichia coli and B. subtilis (Morandi, C et al. (1984) Gene 30:69-77) and a multifunctional plasmid (pME2001) derived from a methanogen capable of replication in a variety of microbial host species (Meile, L. et al., (1985) Bio/Technology 3(1):69-72). Chimeric plasmid vectors, such as pHY460 and pHY310, were constructed from the streptococcal tetracycline resistance (Tc.sup.R) plasmid pAM1 (9.2 kb) and the E. coli vector pACYC177 (3.7 kb). These bifunctional plasmids could replicate and express the Tc.sup.R gene in both E. coli and B. subtilis. Plasmids pHY460 (7.0 kb) and pHY310 (4.8 kb) contained the Tc.sup.R gene of pAM1 and the ampicillin resistance (Ap.sup.R) gene of pACYC177. Both plasmids showed high transformation efficiency in both host cells. pHY460 was maintained stably in B. subtilis. The Pvul, Pstl, BglI and BanI sites in the Ap.sup.R gene and the Hpal, BalI and EcoRV sites in the Tc.sup.R gene can be used for selection of recombinant plasmids by insertional inactivation. In addition, plasmids pHY460 has unique sites for SacII, BstEII, XbaI, AvaI and BamHI. (Ishiwa, H et al., 1984, Gene 32:129-134). Bifunctional vectors (pMH158 and pTO 158) were constructed carrying selective markers and replicons derived from E. coli and S. cerevisiae and containing 21 and 23 unique restriction sites respectively (Heusterspreute, M. et al., (1985), Gene 34:363-366). A vector containing both kanamycin and thiostrepton resistance factors and capable of shuttling between E. coli and Actinomyces has been developed (Biotechnol. Japan Newsservice 3(10:8 (1985)). Other examples include: E. coli-Bacillus shuttle vectors derived from runaway replication plasmids related to Clo DF13 (Andreoli, P. M., (1985) Mol. Gen'l Genet. 199(3):372-380); shuttle vectors suitable for selection of regulatory sequences in B. subtilis and Streptococcus lactis (Van der Vossen, J.M.B.M., (1985) Appl. Environ. Microbiol. 50(2):540-542); a chimeric plasmid capable of shuttling between at least two bacterial species including E. coli, Bacillus or Cornynebacterium (European Patent Application EP 0155594); a chimeric vector containing tetracycline and ampicillin resistance genes and replication origin derived from E. coli and Streptomyces fecalis (European Patent Application 0162725); a shuttle vector capable of transformation of E. coli and Acetobactor aceti (Fukaya, M. et al. (1985) Agric. Biol. Chem. 49(7):2083-2090); a shuttle vector capable transforming both E. coli and Gluconobacter suboxydans (Fukaya, M. et al. (1985) Agri. Biol. Chem. 49(8):2407-2411); a shuttle vector capable of functioning in both Brevibacterium lactofermentum and Cornybacterium glutamicum (Miwa, K. et al. (1985) Gene 39:281-286); chimeric plasmids capable of replicating in E. coli and Saccharomyces cereviriae (PCT Patent Appln. WO 85/05632) and shuttle vectors capable of transforming both Zymomonas mobilis and E. coli (Tonomura, K. et al., (1986) Agric. Biol. Chem. 50(3):805-808).
This invention provides shuttle vectors capable of replication and expression of foreign genetic information in a cyanobacterium and E. coli. More specifically this invention relates to the use of DNA sequences to construct biphasic shuttle vectors for use in the cyanobacterium Agmenellum quadruplicatum strain PR-6 (hereinafter called PR-6), also identified as Synechococcus sp. 7002 (deposited in the American Type Culture Collection as ATCC 27264) and the eubacterium Escherichia coli. This invention also deals with the use of these shuttle vectors to introduce foreign genes into said cyanobacterium, for example the .beta.-galactosidase gene of E. coli. Various aspects of this invention have been disclosed by the inventors in an article in Science 230:805-807 (Nov. 15, 1985) the contents of which are incorporated herein by reference.